Limnol. Oceanogr., 40(l), 1995, , by the American Society of Limnology and Oceanography, Inc.

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1 Limnol. Oceanogr., 40(l), 1995, , by the American Society of Limnology and Oceanography, Inc. Regulation of copper concentration in the oceanic nutricline by phytoplankton uptake and regeneration cycles William G. Sunda and Susan A. Huntsman Beaufort Laboratory, NMFS, NOAA, 10 1 Pivers Island Road, Beaufort, North Carolina Abstract Similar sigmoidal relationships were observed between cellular Cu : C ratios and free cupric ion concentration for the neritic alga Thalassiosira pseudonana and two oceanic species (Thalassiosira oceanica and Emiliania huxleyi) grown in trace metal ion buffered media. Only 5-g-fold variations in cell Cu : C were observed for these species over the [Cu2+] range 3 x lo-l5 to 3 x lo-l2 M, with increasing cell copper vs. [Cu2+] slopes above and below this range. At the mean [Cu2+] for the euphotic zone of the North Pacific ( M), cell Cu : C ratios for the three species were 4.4, 4.4, and 3.8 I.cmol mol-i, similar to values for plankton taken from North Pacific waters. These values also match the mean Cu : C ratio of 4.1 pmol mol -I determined from slopes of linear relationships between Cu and PO, in the nutricline of the central North Pacific and the Redfield C : PO, ratio in plankton of 106 : 1. This agreement provides strong evidence that copper concentrations in remote oceanic nutriclines are regulated by phytoplankton uptake and regeneration processes. It is well established that the concentrations of major nutrients, such as nitrate and phosphate, are controlled by biological uptake and regeneration cycles in seawater. In these cycles, nutrients are taken up by phytoplankton in the euphotic zone and lost to deeper waters with the sinking of biogenic particles such as intact algal cells and zooplankton fecal pellets. The nutrients are then returned to solution with the microbial degradation of the sinking biogenic debris. Such uptake and regeneration cycles lead to marked depletion of nutrients near the surface and enrichment with depth. Increases in nitrate and phosphate concentrations with depth are linearly correlated with one another, and the molar nitrate vs. phosphate slopes (16 : 1) of these correlations approximate the ratios of N : P found in marine plankton. This agreement provides strong evidence that planktonic uptake and regeneration cycles control the distributions of nitrate and phosphate concentrations in the ocean (Redfield et al. 1963). Concentrations of many trace metal micronutrients and nutrient analogs (Zn, Fe, Cu, Ni, and Cd) also increase with depth in the ocean and covary with concentrations of major nutrients (Boyle et al 1976; Bruland 1980; Bruland and Franks 1983; Martin and Gordon 1988). This covariance has led to speculation that concentrations of these metals within the oceanic nutricline are largely regulated by uptake and regeneration cycles similar to those for major nutrients (Morel and Hudson 1984). Quantitative assessment of this hypothesis, however, has been difficult because of uncertainties in free metal ion concentrations in seawater- the primary variables controlling trace metal uptake by phytoplankton (Sunda ; Bruland et al ). This uncertainty resulted pri- Acknowledgments We thank Maria Bondura for technical assistance. This work was supported by a grant from the Oceanic Chemistry Program of the Office of Naval Research. 132 marily from a lack of information about metal complexation by organic ligands. Due to analytical advances, data have recently become available on organic complexation of Cu in seawater, as well as on that of other bioactive metals, such as Zn and Cd (Moffett and Zika 1987; Bruland 1989, 1992). Results from a number of investigations that used different techniques have revealed that copper is heavily complexed in the euphotic zone by a strong organic ligand (or group of ligands) possessing a conditional stability constant of M-l (Coale and Bruland 1988, 1990; Moffett et al. 1990; Sunda and Huntsman 199 1). The ligand concentration generally covaries with that of copper, resulting in a relatively constant free cupric ion concentration of l3 M in coastal and near-surface oceanic waters. In the present study we investigated relationships among external free cupric ion concentration, cellular Cu : C ratios, chlorophyll a, and growth rate for four phytoplankton species. The Cu : C ratios observed in the phytoplankton at typical near-surface free cupric ion concentrations were compared with Redfield ratios of Cu : C derived from relative changes in copper and phosphate concentrations with depth in the oceanic nutricline and the Redfield ratio of C : P in plankton of 106 : 1. Comparisons were also made with Cu : C ratios reported previously for natural plankton samples from Pacific waters. Materials and methods Copper uptake and growth rate studies were conducted with three diatoms [Thalassiosira pseudonana (clone 3H), Thalassiosira weissflogii (clone Actin), and Thalassiosira oceanica (clone 13- l)].and with the coccolithophorid Emiliania huxleyi (clone Al 387). The first two are coastal isolates and the latter two are oceanic. Axenic cultures of these algae were obtained from the Center for the Culture of Marine Phytoplankton, Bigelow Laboratory, and were maintained in f/8 medium (Guillard and Ryther 1962) using sterile technique until needed.

2 Oceanic Cu weelation 133 The methods used in the experiments are comparable to those used in previous algal studies with Mn, Cu, and Zn (SundaandHuntsman 1983,1985,1986,1992). Algal cells were grown at 20 C and ph 8.2&O. 1 in 450-ml polycarbonate bottles containing 200 ml of 36%0 seawater medium. They were grown under fluorescent lighting (Vita-Lite, Duro Test Corp.; 500 bmol quanta m-2 s-l PAR) on a 14 : 10 L/D cycle. Experiments were conducted in enriched natural seawater containing added trace metal ion buffer systems. The experimental seawater was collected from the Gulf Stream with a peristaltic pumping system (Sunda and Huntsman 1983) and was stored for 3.5 yr in the dark at 7 C before use. Media were prepared by passing the seawater through 0.4~pm pore Nuclepore filters and enriching it with 32 PM NaNO,, 2 PM Na,HPO,, 40 PM Na,SiO,, 10 nm Na,SeO,, 0.1 pg liter-l vitamin Br2, 0.1 pg liter- biotin, and 20 yg liter-l thiamin. Trace metal ion buffer systems were added to quantify and control free ion concentrations of Cu and other trace metal nutrients. These buffers consisted of 0.1 mm EDTA, 100 nm FeCl,, 48 nm MnCl,, 100 nm ZnCl,, 40 nm CoCl, 100 nm NiC12, and different concentrations of CuCl,. Copper was added along with an equivalent concentration of EDTA so that its addition at high concentrations would not alter the free EDTA concentration and thereby alter free ion concentrations of other metals. After preparation, the media were equilibrated for 24 h before inoculation of cells. Free ion concentrations of Cu and other trace metal nutrients in the media were computed from the total metal concentration and the extent of metal complexation by EDTA and inorganic ions. Total Cu ranged from 1.O nm to 40 PM as computed from the sum of the measured background concentration and the added concentration of CuC12. Background Cu concentration in the medium (1.O nm) was measured by chemiluminescence analysis (Sunda and Huntsman 199 1). The extent of metal complexation was determined from equilibrium calculations as in previous algal studies in EDTA/metal ion buffer systems (Sunda and Huntsman 1992). The computed ratio of [CuEDTA2-] to [Cu2+] was in the presence of 0.1 mm EDTA, sufficient for the EDTA to well-outcompete typical concentrations (l-3 nm) of strong natural ligands (log K, - 13) that may have been present in the Gulf Stream water when it was collected (Moffett et al. 1990; Sunda and Huntsman 199 1). The logs of the computed free Zn, Co, Mn, and Ni ion concentrations were , , -8.53, and Prior to experiments, cells were transferred from the maintenance medium to experimental medium containing no added copper (log[cu2+] = at the measured background [Cu] of 1.O nm). The cells were preacclimated for a period of 5-9 d (depending on the growth rate) and were then inoculated into experimental media at biomass levels of 0.05-O-2 pmol cell C liter- of medium. The algae were grown for 9-l 1 cell generations to the end of the exponential phase, and total concentrations and volumes of cells were measured daily with a multichannel electronic particle counter (Coulter Counter, model TAII). Table 1. Responses of phytoplankton to copper in EDTAtrace metal ion buffered media. Not detected-nd; not analyzed - NA. -log [cu2+] ND ND , Cell Cell Cu Cell C Chl a Mean Specific km01 (mol (mmol cell vol growth liter- )* liter - I)* liter - I)* &m3) rate (d-l) Thalassiosira oceanica Emiliania huxleyi Thalassiosira weissjlogii ND ND NA ,160 ND ND ,012 ND ,041 ND ND NA ,070 ND ,006 ND ND , Thalassiosira pseudonana 7.6 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA

3 134 Sunda and Huntsman 10 ol A ratio increased to a mean of mol liter- 1 at the three lowest [Cu2+] values (Table 1); each of these ratios was used within its corresponding [Cu2+] range to compute cellular Cu : C ratios in T. oceanica. For T. pseudonana, the cell C : cell volume ratio was 14 mol C liter- l, as measured in parallel cultures at a [Cu2+] of 1 O M. Results Fig. 1. Relationships between cellular Cu: C ratios and log[cu*+] for Thalassiosira oceanica (Cl), Emiliania huxleyi (A), Thalassiosira pseudonana ( x ), and Thalassiosira weissflogii (*). Specific growth rates of cultures were computed from linear regressions of In cell volume vs. time for the exponential phase of growth. Cellular Cu concentrations were measured in exponentially growing cultures, 9-l 0 cell divisions after inoculation. To measure Cu, we filtered cells onto 3-pm-pore Nuclepore filters (1 pm for clone A 1387) and washed them with 3 ml of filtered Gulf Stream water. Filter blanks were prepared by passing the culture filtrates through a second set of filters. The experimental and blank filters were placed in 5-ml Teflon vials and digested at 60 C for 3 d in the presence of 0.4 ml of redistilled HN03. The filter digests were diluted to 4 ml with water from a Milli-Q system (Milli-Q water) and measured for Cu concentration by graphite furnace atomic absorption spectrometry. The cellular Cu concentrations were corrected for filter blanks and divided by the total volume of cells to give cellular Cu concentrations in units of mol Cu per liter of cell volume. Triplicate determinations of cellular Cu at log[cu2+] of for T. pseudonana and for T. oceanica (Table 1) yielded standard deviations about the mean of 4 8 and + 12%. Cellular Cu concentrations were converted to molar Cu : C ratios by dividing them by mean values of experimentally measured cell C : cell volume ratios, determined in either the same cultures at the time of copper measurement (T. oceanica, T. weissflogii, and E. huxleyi; see Table 1) or in separate cultures (T. pseudonana). Cell C was measured by standard 14C techniques after exposing the cells to HCl fumes to remove inorganic C (including CaCO,). Cell volume was measured by Coulter Counter as described above. The mean C : cell volume ratios (&SD) were 19+2 and 8.9kO.4 mol C liter-l for E. huxleyi, and T. weissflogii, and there was no trend in the ratios with variations in [Cu2+] (Table 1). For T. oceanica, the mean C : cell volume ratio was mol liter-l down to a [Cu2+] value of M, but the Measured relationships between cellular Cu : C ratios and free cupric ion concentration are shown in Fig. 1. Cellular Cu : C curves for T. pseudonana, T. oceanica, and T. huxleyi were virtually superimposable over most of the experimental range, while the curve for T. weissflogii deviated downward from the other three. The curves for the first three species had sigmoidal shapes, characterized by broad regions in [Cu2+] ( to 1O-11.5 M) where cellular Cu : C varied by only 5-9-fold despite a 1,OOO-fold variation in free Cu. Above this range, the Cu : C curves for the three species steepened, especially for E. huxleyi. By contrast, the cellular Cu : C curve for T. weissfogii approached those for the other three species at the highest copper level but deviated downward with decreasing cupric ion concentrations and fell below the detection limit for cellular Cu at [Cu2+] < lo-l2 M. For this species, cellular Cu : C was approximately proportional to [Cu2 1 over the measurable experimental range. Copper is an algal nutrient, but it is also toxic at elevated concentrations. Evidence for toxicity, in terms of reduced growth rate, decreased cell chlorophyll, or increased volume per cell, was observed for E. huxleyi, T. weissjlogii, and T. pseudonana at [Cu2+] 1 1 O- 1 M (Table 1). No toxicity was apparent at the highest experimental [Cu2+] for T. oceanica except perhaps for a slight (- 10%) increase in mean volume per cell. No nutritional limitation of growth rate was observed for T. weiss$!ogii, T. pseudonana, or E. huxleyi at the lowest [Cu2+] for these species (lo M), but a slight decrease (5%) was observed at this [Cu2+] for T. oceanica. Cu limitation of T. oceanica was verified in a subsequent experiment in which the lowest [Cu2+] was decreased further by a 5-fold higher concentration of EDTA. In this latter experiment, the growth rate of T. oceanica was decreased by 19% at a [Cu2+] of lo- 15e8 M and by 9% at [Cu2+] of M, relative to the rate at the highest free cupric ion concentration (1 O- 4.2 M) (data not shown). Discussion Cellular Cu accumulation curves-the sigmoidal curves for cellular Cu vs. [Cu2+] for T. pseudonana and the two oceanic species are similar to those for algal accumulation of Zn in the same species (Sunda and Huntsman 1992). Like Cu, Zn is a micronutrient metal which is toxic at elevated concentrations. The intermediate portions of the cellular Zn vs. [Zn +] curves which possess minimum slopes are associated with a negative feedback regulation of cellular Zn uptake by an inducible high-affinity trans-

4 Oceanic Cu regulation 135 port system. Similar negative feedback regulation has been observed for other micronutrient metals (Mn and Fe) in these species (Sunda and Huntsman 1986; Harrison and Morel 1986). Regulation of cellular Cu may also explain the broad intermediate region of the Cu accumulation curves over which there is minimal variation in cell Cu. The region of minimal variation in cell Cu could also be due to saturation of cellular Cu uptake sites. The contrasting near-linear relationship between cellular Cu and [Cu2+] in T. weissflogii suggests that Cu is not regulated in this species. This apparent lack of regulation, and the lack of measurable cellular accumulation at [Cu2+] below lo- l2 M, suggests that Cu is not an essential nutrient for this species, as it is for T. oceanica. Whether it is a required nutrient for the other two species is not known. Regulation of Cu concentrations in the oceanic nutricline by algal uptake and regeneration-results of our study, combined with field observations, support the hypothesis that Cu uptake by phytoplankton and subsequent regeneration at depth control the distribution of Cu in many oceanic nutriclines. Data from depth profiles in the nutricline ( m) of the central North Pacific show consistent linear relationships between Cu and PO, concentrations (Fig. 2). Linear regression analysis of Cu vs. PO4 relationships from four North Pacific profiles reveals high-correlation R2 values ( ) and consistent Cu : PO4 slopes of mmol mol-1 (Table 2). If the observed linear Cu vs. PO4 relationships are controlled by phytoplankton uptake and regeneration, then the ACu/APO, slopes in the nutricline should equal the ratios in which these elements occur in phytoplankton cells. According to this reasoning and the above linear regression slopes, the mean ratio of Cu : P in North Pacific phytoplankton should equal 0.44 f mmol mol- l (Table 2). If we assume a C : PO, ratio of 106 : 1 (the classic Redfield ratio), then we compute a Cu : C ratio for the North Pacific plankton of 4.1 pmol mol-i. This Redfield Cu : C ratio is consistent with the value we would predict from recent measurements of free cupric ion concentration in North Pacific seawater and the measured relationships between Cu : C and [Cu2+] in our oceanic algal isolates (T. oceanica and E. huxleyi) and in the coastal species T. pseudonana. Coale and Bruland (1990) recently measured profiles of free cupric ion concentration and organic Cu complexation at the same stations where Martin et al. (1989) determined nutricline profiles for Cu and PO4 concentrations. They found that Cu was highly complexed by organic ligands within the euphotic zone and that [Cu2+] varied little within this layer from one station to next. Free cupric ion concentration in the euphotic zone varied from 1 O to 1 O- 12m7 M, with a mean value of - 1 O M. That value falls near the middle of the broad plateau region of the cellular Cu : C curves for T. oceanica, E. huxleyi, and T. pseudonana, where there is minimum change in cellular Cu with variations in [Cu2+]. At that [Cu2+], these three species had Cu : C ratios of 4.4, 3.8, and 4.4 pmol mol-i, consistent with the above mean Redfield value of 4.1 bmol mol- 1 deter- I _ E E - s 3-2- l- 8 m Fig. 2. A. Cu vs. PO, relationships for depth profiles in the central North Pacific (A-39.6 N, W; 20-1,500 m; Martin et al. 1989), the Southern Ocean m-60.8 S, 63.4 W; 30-1,850 m; Martin et al. 1990), and the North Atlantic (El-47 N, 2O W; 20-2,900 m; Martin et al. 1993). B. Cu vs. PO, relationships for depth profiles from the central North Pacific (A-32.7 N, 145.O W; O-4,875 m) and two stations off the central California coast m--37.0, W; 25-3,900 m; Cl-36.9 N, W; 1 O-2,250 m). (Data in panel B from Bruland 1980.) In all cases, data from within the nutricline are represented by the lower portions of curves to the left of the sharp break in slope. mined from Cu vs. PO4 regressions in the nutriclines of North Pacific stations. Cu : C ratios of hmol mol- are also consistent with those for actual net plankton samples collected from near-surface Pacific waters (Table 3), providing additional strong evidence that plankton uptake and regeneration

5 136 Sunda and Huntsman Table 2. Regression slopes of Cu : PO, and resulting Cu : C ratios, based on depth-dependant variations in dissolved copper and phosphate in oceanic nutriclines. Location North Pacific 32.7 N, 145.O W 39.6 N: W 45.O N, W 50.0 N, 145.O W North Atlantic 47 N, 2O W Drake Passage 60.8 S, 63.4 W Depth b-0 O O O O O * Based on Redfield C : P ratio of 106 : 1. ACu/APO, cu:c* (mmol mol I) (pm01 mol-i) R* n Reference Bruland Martin et al Martin et al Martin et al Martin et al Martin et al regulate nutricline Cu concentrations. For example, the mean Cu : P ratio was O mmol mol- in a set of 2 1 net (50~pm mesh) plankton samples collected in transects running from - 50 to 1,100 km off the coasts of southern California and northern Mexico (Martin et al 1976). This value converts to a mean Cu : C of 4.6k2.3 hmol mol- 1 if we once again assume a C : P ratio of 106 : 1. Much of the large standard deviation about the mean results from two unusually high values; if we exclude these, we obtain a mean Cu : C ratio for the remaining 19 samples of pm01 mol-l. Linear relationships between Cu and PO, concentrations in the nutricline are also found in other remote oceanic locations, including the North Atlantic and Southern Ocean (Fig. 2A; Table 2). For these two locales, we compute Redfield Cu : C ratios of 2.8 and 6.4 pmol mol-i, respectively, from Cu vs. PO4 regressions and a C : P ratio of 106 : 1. These values bracket those found in the central North Pacific and fall within the range of values found for natural plankton samples (Table 3). Slight differences in Cu : P relationships within oceanic nutriclines may reflect local differences in free cupric ion concentration or species composition of plankton. We note that a Cu : C ratio of 2.8 pmol mol- would occur in our two oceanic phytoplankton species (T. oceanica and E. huxleyi) at free cupric ion concentrations of 10-13a7 and M and a ratio of 6.5 pmol mol-1 would occur at and lo M. These values are consistent with the range in [Cu ] measured previously in open ocean waters of the North Atlantic and Pacific Oceans (Coale and Bruland 1990; Moffett et al. 1990). Although phytoplankton uptake and regeneration ap- pear to dominate Cu cycling in the nutriclines of most remote oceanic regions, other processes such as inputs of Cu from aeolean and riverine sources and scavenging by nonbiogenic particulates (e.g. Mn and Fe oxyhydroxides) become increasingly important as one approaches the continents. In stations sampled by Bruland (1980) off the central California coast, such processes mask the effects of biogenic cycling, resulting in a lack of correlation between copper and phosphate within the nutricline (Fig. 2B). In regions where there are intense aeolean inputs from deserts, such as the northwestern Indian Ocean, distinct surface maxima are observed in Cu concentrations (Saager et al. 1992). Redfield-type behavior also is not observed below the nutricline in the world oceans. Below the phosphate maximum at roughly 1,000 m in the North Pacific, Cu concentrations continue to increase with depth as phosphate concentrations decrease, resulting in negative correlations between the two (Fig. 2B). The increase in Cu concentrations with depth in the deep sea has been attributed to difhtsion of this metal from Cu-rich bottom sediments combined with midwater scavenging by unknown particulate phases (Boyle et al. 1977; Bruland 1980). The marked difference in Cu behavior between the nutricline and deeper waters in the Pacific may reflect large differences in the ventilation age of the water-the time since it last resided at the surface and was subject to surface processes, such as algal growth. Water in the nutricline (upper 800 m) of the North Pacific contains 14C released by hydrogen bomb testing during the early 1960s (Williams and Druffel 1987). Thus, this water is relatively young and has recently experienced near-surface biogenic Table 3. Cu : PO, and resultant estimated Cu : C ratios in natural plankton samples from the Pacific. Location n Cu : PO, (mmol mol- I) cu:c* (Ccmol mol- I) Reference Equatorial Pacific kO Collier and Edmond 1983 Pacific off S. California and N. Mexico f Martin et al * Based on assumed C : PO, of 106 : 1.

6 Oceanic Cu regulation 137 scavenging. By contrast, intermediate and deep waters of the North Pacific have estimated ages of - 1,000 yr. Conclusions We observed a close agreement among estimated Cu : C ratios in oceanic plankton obtained from three entirely different sources: measured relationships between cellular Cu : C ratios in algal cultures as functions of cupric ion concentration combined with recent measurements of free cupric ion concentrations in near-surface North Pacific seawater; measurements of Cu concentrations in plankton samples from the Pacific Ocean; and slopes of Cu vs. phosphate relationships in the nutricline of the North Pacific combined with the Redfield C : PO, ratio in plankton. This agreement shows a basic coherence between our laboratory data and the various field measurements. It also provides strong evidence that phytoplankton uptake and regeneration represent the dominant processes regulating copper concentrations in the nutriclines of remote oceanic regions. References BOYLE, E. A., F. R. SCLATER, AND J. M. EDMOND On - the marine geochemistry of cadmium. Nature 263: The distribution of dissolved copped: the Pacific. Earth Planet. Sci. Lett. 37: BRULAND, K. W Oceanographic distributions of cadmium, zinc, nickel and copper in the North Pacific. Earth Planet. Sci. Lett. 47: Complexation of zinc by natural organic ligands in-the central North Pacific. Limnol. Oceanogr. 34: Complexation of cadmium by natural organic ligands in the central North Pacific. Limnol. Oceanogr. 37: , J. R. DONAT, AND D. A. HUTCHINS Interactive influences of bioactive trace metals on biological production in oceanic waters. Limnol. Oceanogr. 36: 1555-l , AND R. P. FRANKS Mn, Ni, Cu, Zn, and Cd in the western North Atlantic, p In C. S. Wong et al. [eds.], Trace metals in sea water. Plenum. COALE, K. H., AND K. W. BRULAND Copper complexation in the northeast Pacific. Limnol. Oceanogr. 33: ,AND Spatial and temporal variability in copper complexation in the North Pacific. Deep-Sea Res. 34: COLLIER, R. W., AND J. M. EDMOND Plankton compositions and trace element fluxes from the surface ocean, p In C. S. Wong et al. [eds.], Trace metals in sea water. Plenum. GUILLARD, R. R. L., AND J. H. RYTHER Studies of marine planktonic diatoms. 1. CycZoteZZa nana Hustedt and Detonula confervacea (Cleve) Gran. Can. J. Microbial. 8: HARRISON, G. I., AND F. M. M. MOREL Response of the marine diatom Thalassiosira weissflogii to iron stress. Limnol. Oceanogr. 31: MARTIN, J.H.,K.W. BRULAND,AND W.W. BROENKOW Cadmium transport in the California Current, p. 159-l 84. In H. L. Windom and R. A, Duce [eds.], Marine pollutant transfer. Heath. ---,S. E. FITZWATER, R. M. GORDON, C.N. HUNTER, AND S. J. TANNER Iron, primary production and carbonnitrogen flux studies during the JGOFS North Atlantic bloom experiment. Deep-Sea Res. 40: 115-l 34. -, AND R. M. GORDON Northeast Pacific iron distributions in relation to phytoplankton productivity. Deep-Sea Res. 35: AND S. E. FITZWATER Iron in antarctic waters. Nature 345: 156-l ,AND W.W. BROENKOW VER- TEX: Phy;oplankton/iron studies in the Gulf of Alaska. Deep-Sea Res. 36: MOFFETT, J. W., L. E. BRAND, AND R. G. ZIKA Distribution and potential sources and sinks of copper chelators in the Sargasso Sea. Deep-Sea Res. 37: AND R. G. ZIKA Solvent extraction of copper a&ylacetonate in studies of copper(i1) speciation in seawater. Mar. Chem. 21: 30 l MOREL, F. M. M., AND R. J. M. HUDSON The geo- biological cycle of trace elements in aquatic systems: Redfield revisited, p In W. Stumm led.], Chemical processes in lakes. Wiley. REDFIELD, A. C., B. H. KJZTCHUM, AND F. A. RICHARDS The influence of organisms on the composition of seawater, p In M. N. Hill [ed], The sea. V. 2. Wiley. SAAGER, P. M., H. J. W. DE BAAR, AND R. J. HOWLANLL Cd, Zn, Ni and Cu in the Indian Ocean. Deep-Sea Res. 39: SUNDA, W. G. 1988/1989. Trace metal interactions with marine phytoplankton. Biol. Oceanogr. 6: ,AND S.A. HUNTSMAN Effect of competitive interactions between manganese and copper on cellular manganese and growth in estuarine and oceanic species of the diatom Thalassiosira. Limnol. Oceanogr. 28: ,AND Regulation of cellular manganese and manganese transport rates in the unicellular alga Chlamydomonas. Limnol. Oceanogr. 30: 7 l-80. -,AND Relationships among growth rate, cellular manganese concentrations, and manganese transport kinetics in estuarine and oceanic species of the diatom Thalassiosira. J. Phycol. 22: ,AND The use of chemiluminescence and ligand competition with EDTA to measure copper concentration and speciation in seawater. Mar. Chem. 36: ,AND Feedback interactions between zinc and phytoplankton in seawater. Limnol. Oceanogr. 37: WILLIAMS, P. M., AND E. R. M. DRUFFXL Radiocarbon in dissolved organic matter in the central North Pacific Ocean. Nature 330: Submitted: 4 January 1994 Accepted: 25 July 1994 Amended: 23 August 1994